Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Bone resorbing activities of 24-hydroxy stereoisomers of 24-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

R and S isomers of 24-OH-D₃ and 24,25-(OH)₂D₃ were tested for their effects on bone resorption $\text{in vitro}$. 24(R), 25-(OH)₂D₃ was more active than 24(S),25-(OH)₂D₃. Likewise, 24(R)-OH-D₃ was more active than 24(S)-OH-D₃. The bone resorbing activity of 24(R)-OH-D₃ was equivalent to that of 25-OH-D₃; 24(R),25-(OH)₂D₃ was somewhat less potent. The results indicate that there is discrimination between the isomers of these compounds at the level of the responding tissue.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Behavioral control by striatal adenosine A2A‐dopamine D2 receptor heteromers

G protein‐coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A_2A receptors (A_2AR) and dopamine D₂ receptors (D₂R) predominantly form A_2AR‐D₂R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A_2AR and D₂R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain‐related differences, a new D₂R‐deficient mouse with the same genetic background (CD‐1) than the A_2AR knock‐out mouse was generated. Locomotor activity, pre‐pulse inhibition (PPI) and drug‐induced catalepsy were then evaluated in wild‐type, A_2AR and D₂R knock‐out mice, with and without the concomitant administration of either the D₂R agonist sumanirole or the A_2AR antagonist SCH442416. SCH442416‐mediated locomotor effects were demonstrated to be dependent on D₂R signaling. Similarly, a significant dependence on A_2AR signaling was observed for PPI and for haloperidol‐induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A_2AR‐D₂R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders.     

Effects of 1,25(OH)2D3 and vitamin D receptor on peripheral CD4+/CD8+ double‐positive T lymphocytes in a mouse model of systemic lupus erythematosus

This study aims to explore effects of 1,25(OH)₂D₃ and vitamin D receptor (VDR) on peripheral CD4⁺/CD8⁺ double‐positive (DP) T lymphocytes in systemic lupus erythematosus (SLE). MRL‐LPr/LPr mice with SLE (n = 20) and normal MRL mice (n = 20) were assigned into the control group (normal mice, without feeding with 1,25(OH)₂D₃), the 1,25(OH)₂D₃ group (SLE mice, feeding with 1,25(OH)₂D₃), the VDR‐knock‐in + 1,25(OH)₂D₃ group (SLE mice, VDR‐knock‐in, feeding with 1,25(OH)₂D₃) and the VDR‐knockout group (normal mice, VDR‐knockout, without feeding with 1,25(OH)₂D₃) (n = 10 per group). Levels of T lymphocytes were measured by flow cytometry. The mRNA and proteins expressions of inflammatory factors were measured by qRT‐PCR and ELISA. Extracellular signal‐regulated kinase‐1/2 (ERK1/2) expression was measured by Western blotting. Compared with normal mice, SLE mice showed reduced levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes. The levels of SLE‐related indicators all increased significantly, followed with severe skin ulcers and urinary system infection. With the increase in time, skin ulcers and urinary system infection were significantly improved, levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes increased, and levels of SLE‐related indicators all decreased in the 1,25(OH)₂D₃ group. There were no significant changes in bioindicators in the control and the VDR‐knock‐in + 1,25(OH)₂D₃ groups. The symptoms of SLE gradually occurred in the VDR‐knockout group. This study demonstrates that VDR and 1,25(OH)₂D₃ could elevate CD4⁺/CD8⁺ DP T lymphocytes and reduce expressions of inflammatory factors, thus inhibiting the development and progression of SLE.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

Osteoclast‐specific inactivation of the integrin‐linked kinase (ILK) inhibits bone resorption

Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the α_vβ₃ integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin‐linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast‐specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP‐Cre transgenic mice. The TRAP‐Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast‐specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast‐specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C‐terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the β₃ integrin gene were inactivated (ILK^+/?; β) also had increased trabecular thickness, confirming that β₃ integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts. J. Cell. Biochem. 110: 960–967, 2010. © 2010 Wiley‐Liss, Inc.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Disrupted‐in‐schizophrenia 1 functional polymorphisms and D2/D3 receptor availability: A [11C]‐(+)‐PHNO imaging study

The disrupted‐in‐schizophrenia 1 (DISC1) protein has been implicated in a range of biological mechanisms underlying chronic mental disorders such as schizophrenia. Schizophrenia is associated with abnormal striatal dopamine signalling, and all antipsychotic drugs block striatal dopamine 2/3 receptors (D_2/3Rs). Importantly, the DISC1 protein directly interacts and forms a protein complex with the dopamine D₂ receptor (D₂R) that inhibits agonist‐induced D₂R internalisation. Moreover, animal studies have found large striatal increases in the proportion of D₂R receptors in a high affinity state (D₂^highR) in DISC1 rodent models. Here, we investigated the relationship between the three most common polymorphisms altering the amino‐acid sequence of the DISC1 protein (Ser704Cys (rs821616), Leu607Phe (rs6675281) and Arg264Gln (rs3738401)) and striatal D_2/3R availability in 41 healthy human volunteers, using [¹¹C]‐(+)‐PHNO positron emission tomography. We found no association between DISC1 polymorphisms and D_2/3R availability in the striatum and D₂R availability in the caudate and putamen. Therefore, despite a direct interaction between DISC1 and the D₂R, none of its main functional polymorphisms impact striatal D_2/3R binding potential, suggesting DISC1 variants act through other mechanisms.     

Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor,modulating mammary epithelial cell growth

The vitamin D₃ receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D₃ synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D₃‐induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α‐hydroxylase, 1α‐OHase), the enzyme that generates the biologically active VDR ligand, 1α,25‐dihydroxyvitamin D₃ (1,25D₃), and CYP24 (24‐hydroxylase, 24‐OHase), a VDR‐1,25D₃ induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25‐hydroxyvitamin D₃ (25D₃) to the active ligand, 1,25D₃, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co‐cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D₃ or 1,25D₃ compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D₃ to 1,25D₃, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D₃‐induced growth regulation of ductal epithelium. J. Cell. Biochem. 112: 3393–3405, 2011. © 2011 Wiley Periodicals, Inc.     

Deep‐brain three‐photon microscopy excited at 1600 nm with silicone oil immersion

Three‐photon microscopy excited at the 1700‐nm window (roughly covering 1600‐1840 nm) is especially suitable for deep‐brain imaging in living animals. To match the brain refractive index, D₂O has been exclusively used as the immersion medium. However, the hygroscopic property of D₂O leads to a decrease of transmittance of the excitation light and as a result a decrease in three‐photon signals over time. Solutions such as replacing D₂O from time to time, wrapping both the objective lens and the immersion D₂O, and sealing D₂O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D₂O for deep‐brain imaging. Excited at 1600 nm, our comparative deep‐brain imaging using both D₂O and silicone oil immersion show that silicone oil immersion yields 17% higher three‐photon signal in third‐harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three‐photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long‐span three‐photon brain imaging excited at the 1700‐nm window.